Ferulic Acid Antagonized The Transdifferentiation Of Renal Tubular Epithelial Cells To Myofibroblast

Objective: To investigate the effect of ferulic acid on the transdifferentiation of human renal tubular epithelial cells(HK-2)into myofibroblasts.Methods: Using HK-2 as the research object,cells were stimulated with different concentrations(0,1,5,and 10 ng/mL)of TGF-?1 for different times(12,24,48,and 72 h).The optimal conditions for establishing renal tubular epithelial cells transdifferentiated myofibroblasts(model cells)were screened by observing cell morphology and ?-smooth muscle actin(?-SMA)fluorescence intensity.namely the concentration and time of TGF-?1.Screening to establish a model of transdifferentiation of renal tubular epithelial cells into myofibroblasts.Different concentrations of ferulic acid(125 ?mol/L,250 ?mol/L,500 ?mol/L)were used to treat the model cells.The effects of ferulic acid on the morphology of the model cells were observed under microscope,and the fibronectin(Fn)and type I collagen(Col I)proteins in the cell supernatant were detected by enzyme-linked immunosorbentassay(ELISA)method.The expression level of ?-SMA and integrin-linked kinase(ILK)protein in the cells was detected by Western blotting.The mRNA levels of ?-SMA,Fn,Col I,and ILK were detected by real-time fluorescent quantitative(qPCR).Results:(1)10 ng/ml TGF-?1 intervened cells for 24 h,Cell morphology changes from elliptical or polymorphous to long shuttle,The morphological change was most obvious,and the ?-SMA protein positive signal was the strongest.(2)The effect of ferulic acid on the morphology of the model cells: the control group cells were oval or polygonal;the model group cells were long shuttle-shaped;the low-,medium-,and high-dose ferulic acid group,the cell morphology tends to the control group.(3)The effect of ferulic acid on the expression of ?-SMA,Fn,Col I,ILK and ?-SMA proteins in model cells: Immunofluorescence was used to detect the positive signal of ?-SMA protein in each group of cells.The positive signal in model group was the strongest,low,medium,and high.The ?-SMA protein positive signal of high-dose ferulic acid group cells decreased with the increase of ferulic acid concentration.The expression levels of Fn and Col I protein in supernatants of the cells were detected by ELISA: the expression levels of Fn protein in the control group,model group,low-dose ferulic acid group,middle-dose ferulic acid group,and high-dose ferulic acid group were(138.57±14.99).Pg/mL,(554.82±7.83)pg/mL,(491.16±9.69)pg/mL,(270.23±18.47)pg/mL,(195.99±5.91)pg/mL(P<0.01),Col I Protein Expression The levels were(1418.60±154.08)pg/mL,(6652.40±458.53)pg/mL,(5430.77±227.28)pg/mL,(4424.77±329.92)pg/mL,(2279.07±369.12)pg/mL(P< 0.01).The expression levels of ?-SMA and ILK protein in the cells were detected by Western blot: The levels of ?-SMA protein expression in the control group,the model group,the low-dose ferulic acid group,the middle-dose ferulic acid group,and the high-dose ferulic acid group were0.14±0.01,0.40±0.01,0.36±0.01,0.23±0.01,0.16±0.01(P<0.01),respectively.The ILK protein expression levels were 0.14±0.02,0.46±0.02,0.38±0.01,0.33±0.01,and 0.29±0.01(P<0.01),respectively.(4)Effects of ferulic acid on the expression of ?-SMA,Fn,Col I and ILK mRNA in model cells: control group,model group,ferulic acid low dose group,ferulic acid medium dose group and ferulic acid high dose group?-The expression of SMA mRNA was 1.00±0.00,1.72±0.14,1.38±0.10,1.20±0.08,and 1.00±0.08(P<0.01),respectively.The expression of Fn mRNA was 1.00±0.00,2.96±0.21,1.95±0.15,1.69±0.13,1.26±0.05(P<0.01),respectively.Col I mRNA expression levels were 1.00 ± 0.00,2.96 ± 0.12,2.62 ± 0.05,1.79 ± 0.07,and 1.17 ± 0.04(P <0.01),respectively.ILK mRNA expression levels were 1.00±0.00,2.31±0.16,1.78±0.11,1.55±0.11,1.29±0.09(P<0.01),respectively.(5)Correlation analysis of ILK protein expression with Fn,Col I,and ?-SMA proteins:ILK was positively correlated with Fn,Col I,and ?-SMA,and thecorrelation coefficients were 0.897(P<0.05),0.938(P<0.05),0.890(P <0.05),respectively.Conclusion:(1)10 ng/mL TGF-?1 stimulation of HK-2 cells for 24 h is a better condition for transdifferentiation of HK-2 cells into myofibroblasts induced by TGF-?1.(2)Ferulic acid downregulated the mRNA and protein expression levels of ILK,Fn,Col I,and ?-SMA in the model group,and inhibited the transdifferentiation of HK-2 cells into myofibroblasts induced by TGF-?1.(3)The effect of ferulic acid inhibition on the transdifferentiation of hk-2 cells into myofibroblasts may be related to the inhibition of the downstream signaling factor ILK of TGFbeta 1.